C. elegans, we identified splicing factor RNP-6/PUF60 whose activity suppresses immunity, but promotes longevity, suggesting a tradeoff between these processes. Bacterial pathogen exposure affects gene expression and splicing in a rnp-6 dependent manner, and rnp-6 gain and loss-of-function activities reveal an active role in immune regulation. Another longevity promoting splicing factor, SFA-1, similarly exerts an immuno-suppressive effect, working downstream or parallel to RNP-6. RNP-6 acts through TIR-1/PMK-1/MAPK signaling to modulate immunity. The mammalian homolog, PUF60, also displays anti-inflammatory properties, and its levels swiftly decrease after bacterial infection in mammalian cells, implying a role in the host response. Altogether our findings demonstrate an evolutionarily conserved modulation of immunity by specific components of the splicing machinery.Splicing is a vital cellular process that modulates important aspects of animal physiology, yet roles in regulating innate immunity are relatively unexplored. From genetic screens in  Caenorhabditis elegans have provided significant and unique insights into underlying mechanisms of innate immunity (Innate immunity comprises the first line of defense against invading micro-organisms. The presence of pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs) activates conserved signaling pathways to protect the host and bring infection under control . Over thimmunity . Pioneerimmunity , HLH-30/immunity  and β-caimmunity  regulateC. elegans, the MAPK cascade of NSY-1-SEK-1-PMK-1 is essential to innate immunity, and has orthologs in mammals (ASK1 MAPKKK-MKK3/6 MAPKK-p38 MAPK pathway) playing a similar role  domain protein and an ortholog of mammalian SARM   . VariousC. elegans, including heavy metal resistance, metabolism and lifespan  are removed from primary transcripts, and protein coding sequences (exons) are joined together to form the mature mRNA. This activity is catalyzed by a family of specialized proteins called splicing factors . Differelifespan , yet howC. elegans wildtype N2 animals  Binding Splicing Factor 60 (PUF60). PUF60 facilitates the association of the U2 snRNP with target RNA molecules  was highly resistant to cold stress (dh1127) was created using CRISPR gene editing, which showed the same phenotype, confirming the casual relation  mutant with different pathogenic bacteria, namely Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa. Unexpectedly, this mutant was more sensitive to all pathogenic bacteria tested (dh1125) (S. aureus plates. The rnp-6(dh1127) mutant displayed the same sensitive phenotype (rnp-6(dh1127) animals were more sensitive to S. aureus, we did not observe a difference in bacterial burden  mutant showed robust longevity at 20°C  . We alsohenotype , suggestl burden . Interes at 20°C  and a no at 20°C , indicat at 20°C . Taken tdh1188). We performed qRT-PCR and western blots to detect the endogenous levels of RNP-6 at both transcript and protein levels. However, no significant changes could be detected after S. aureus infection  under both non-infected (1483 genes)   and infe3 genes) . Under b3 genes) . 662 gennfection   and lysozyme (ilys-2) was severely compromised by rnp-6 G281D substitution (rnp-6 (nhr-62 and nhr-3) and metabolic enzymes (cth-1 and fat-5) was refractory to the repression by infection in the rnp-6 G281D mutant  mutants, we found a larger number of alternatively spliced genes (456 genes) (S. aureus could be underestimated as a result of the short infection time used (4 hr post-infection) and tissue-specific alternative splicing masked in the transcriptome from whole animals. Over half of the genes (43 out of 83) (rnp-6(dh1127) mutants when compared to infected wildtype N2 animals. This suggests that the splicing alteration induced by infection is partly dependent on rnp-6. We also analyzed the data with the SAJR algorithm, which gives a detailed classification of the splicing events (S. aureus infection induced splicing events were predominated by ‘cassette exon’ (44.9%) and ‘retained intron’ (22.4%). This was also true when we compared infected rnp-6(1127) mutants with infected wildtype animals   mutants  . The numt of 83)  showing g events . In wild’ 15.2%) . Using R mutants . Infecti mutants . Further animals . Intrigunfection , suggestrnp-6 compromises immunity . Furthermore, overexpressing RNP-6 greatly compromised infection survival upon S. aureus    to inveset genes . Among tet genes , 17 wereet genes . For thetitution , 55 of tn mutant . These rrnp-6 and pmk-1. Knocking down rnp-6 increased the animals’ survival upon S. aureus infection, and deletion of pmk-1 or its upstream activator tir-1 completely abolished the beneficial effect of rnp-6 reduction , area under survival curves)  . Overexpckground . The rnpnditions . We concpmk-1 and rnp-6, we next sought to test whether RNP-6 affects the activity of PMK-1 by measuring phospho-PMK-1. Upon RNAi knockdown of rnp-6, we observed a significant increase in phospho-PMK-1  animals under non-infected or S. aureus infected conditions  and RAW264.7 macrophage-like cells with S. aureus and observed a sharp reduction of PUF60 protein levels in all three cell types    without n 24 hr) . EnhanceC. elegans, bacterial infections affect mRNA splicing, and perturbations of splicing is a trigger for immune responses while, by inference, increased splicing activity compromises immunity, thus providing a new explanation for how immune responses are modulated in the worms. Importantly, the function of RNP-6/PUF60 in immunity is conserved in mammalian cells. Thus, our results also shed light on the mechanisms of human diseases that involve PUF60.Our data reveal that the splicing factor RNP-6/PUF60 is a novel regulator of immunity and lifespan. Viruses commonly interact with the host splicing machinery, whereas not much is known regarding bacterial interference with host RNA splicing . We provrnp-6 G281D substitution and overexpression of splicing factors (RNP-6 and SFA-1) lead to sensitivity to infection but promote longevity. Notably, the rnp-6 G281D mutant is resistant to abiotic stresses, suggesting the sensitivity towards infection is mediated by specialized mechanisms rather than a general decline of fitness in this mutant.Long-lived animals are often more stress resistant. Stress resistance and longevity are thought to be tightly and positively correlated, but this idea has been recently disputed . The behrnp-6 G281D mutation. In the context of survival, phenotypically this mutation appears to be a gain of function as both the G281D substitution and RNP-6 over-expression lead to compromised infection survival but prolonged lifespan on OP50. However, the G281D substitution also shows characteristics of a loss of function mutation in other aspects. For example, the transcriptional and splicing changes induced by the G281D substitution are complex with some genes showing patterns similar to that of rnp-6 RNAi while others having different patterns. We believe that the G281D substitution does not simply increase or decrease the activity of RNP-6 but rather causes a complex change in the affinity of the RNA binding domain to different RNA molecules. The location of the substitution in the RNA binding motif also supports this idea.One question that remains to be investigated is the nature of the C. elegans animals is that the changes observed might be due to changes in isoform expression pattern in different tissues rather than alternate splicing within the same tissue, as genes can have tissue enriched isoforms. Nevertheless, the rnp-6 dependence of these splicing changes argues in favor of a true splicing effect.One limitation of the splicing assaying with PCR on RNA extracted from whole In mammals, PUF60 is also known as far-upstream element binding protein, which inhibits the transcriptional factor FUSE binding protein . AlthougC. elegans is not fully understood  of splicing . Unlike ongevity . Aberran elegans . Also, inhibited . One attrnp-6 levels remain stable after infection in C. elegans, infection reduces the abundance of PUF60 in various mammalian cells. We hypothesize that infection induced down-regulation of PUF60 might be a recent evolutionary adaptation of the immune system. Alternatively, the activities of RNP-6 may be regulated in C. elegans by other ways without affecting its abundance. Indeed, infection induced splicing remodeling is dependent on RNP-6, and knocking-down rnp-6 affects splicing of genes whose splicing is regulated by infection, suggesting that RNP-6 activity is influenced by infection. Reduction of PUF60 by RNAi is sufficient to induce pro-inflammatory cytokine expression, suggesting that PUF60 reduction induced by bacterial infection is a trigger of inflammation. This intriguing observation raises the question of how perturbation of splicing can activate immune responses. One potential explanation is aberrant splicing may produce immuno-stimulatory RNAs which can be detected by nucleic acid sensing PRRs, such as RIG-I  is/are involved? How does the G281D mutation change the molecular functions of RNP-6/PUF60? Further studies are needed to precisely elucidate how splicing affects host responses towards infection.rnp-6(dh1124), rnp-6(dh1125), rnp-6(dh1127), hlh-30(tm1978), rnp-6(dh1188), rnp-6(dh1127); wbmEx340[eft-3p::3xFLAG::sfa-1 cDNA::unc-54 3'UTR]; [myo-3p::mCherry::unc-54 3'UTR], dhEx1132 (RNP-6 OE#1), dhEx1133 (RNP-6 OE#2), dhEx1134 (RNP-6 OE#3), pmk-1(km25), tir-1(tm3036), pmk-1(km25); dhEx1132, rnp-6(dh1127); pmk-1(km25), daf-16(mu86), wbmEx340[eft-3p::3xFLAG::sfa-1 cDNA::unc-54 3'UTR]; [myo-3p::mCherry::unc-54 3'UTR], rnp-6(dh1188); wbmEx340[eft-3p::3xFLAG::sfa-1 cDNA::unc-54 3'UTR]; [myo-3p::mCherry::unc-54 3'UTR] and pmk-1(km25); wbmEx340[eft-3p::3xFLAG::sfa-1 cDNA::unc-54 3'UTR]; [myo-3p::mCherry::unc-54 3'UTR]. The animals were maintained at 20°C following standard procedures (The following strains were used in this study: N2 (wildtype), ocedures . For allHeLa and MEF cells were obtained from ATCC. RAW264.7 cells were obtained from ECACC. The cells were cultured at 37°C with 5% carbon dioxide in a humid atmosphere. Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) was used as the culturing medium. The cells were tested and are negative for mycoplasma contamination.Staphylococcus aureus (strain MW2), Enterococcus faecalis (strain ATCC 29212), Pseudomonas aeruginosa (strain PA14) and Escherichia coli  were used in this study. Bacteria were maintained in glycerol stocks at −80°C. When needed, bacteria were streaked out on appropriate agar plates. Tryptic soy agar (TSA) plates for S. aureus, brain heart infusion (BHI) plates for E. faecalis and lysogeny broth (LB) plates for P. aeruginosa and E. coli. Colonies were picked into designated growth medium. S. aureus was grown in tryptic soy broth (TSB) medium. E. faecalis was grown in BHI medium. P. aeruginosa andE. coliwere grown in LB medium. 100 μg/mL of ampicillin was supplemented to the plates and cultures of Escherichia coli (strain HT115 and OP50 xu363) to maintain the plasmids for RNAi feeding experiments.E. coli OP50. The cold resistant segregants were then pooled together, and genomic DNA were purified using Gentra Puregene Kit (Qiagen). The pooled DNA was sequenced on an Illumina HiSeq platform (paired-end 150 nucleotide). MiModD software (http://www.celegans.de/en/mimodd) was used to locate the mutations.The cold resistant mutants were crossed with Hawaiian CB4856 males. Young adults of the F2 generation were exposed to cold stress, and the survivors were singled and propagated on standard nematode growth medium (NGM) plates seeded with For all stress assays, synchronized young adult hermaphrodites were used. For cold resistance, worms were incubated at 2°C on standard NGM plates with OP50 for the indicated time. The animals were then allowed to recovered at 20°C for 12–24 hr. After that, their survival was score. For heat resistance, animals on standard NGM plates seeded with OP50 were transferred to 35°C. Survival was monitored every hour. For oxidation stress, worms were placed on NGM plates with OP50 supplemented with 20 mM paraquat. Survival was monitored every 12 hr. Animals that did not respond to gentle touch by a worm pick were interpreted as dead.S. aureus (Strain MW2), E. faecalis (Strain ATCC 29212) and P. aeruginosa (Strain PA14) were grown in the appropriate medium at 37°C with gentle shaking overnight. 20 μl of the overnight bacterial cultures was seeded at the center of the relevant agar plates, TSA plates with 10 μg/mL nalidixic acid (NAL) for S. aureus, BHI plates with 10 μg/mL NAL for E. faecalis and modified NGM (Peptone content 3.5 g/L instead of 2.5 g/L), for P. aeruginosa. For full lawn experiments, 100 μl of the bacterial culture was seeded and spread all over the surface of the agar plate. The seeded killing assay plates were allowed to grow overnight at 37°C. On the next day, the plates were left at room temperature for at least 6 hr before the infection experiments. Around 25 synchronized young adult worms were transferred to the plates. three technical replicate plates were set up for each condition. The plates were then incubated at 25°C unless specifically stated. Scoring was performed every 24 hr for E. faecalis or every 12 hr for S. aureus and P. aeruginosa. Worms were scored as dead if the animals did not respond to gentle touch by a worm pick. Worms that crawled off the plates or had ruptured vulva phenotypes were censored from the analysis. All C. elegans killing assays were performed three times independently unless otherwise stated. Genotypes were concealed for all C. elegans infection survival experiments in order to eliminate any investigator-induced bias. Results of each biological replicate of infection survival and lifespan experiments can be found in Animals were sterilized with treatment of 5-Fluoro-2'-deoxyuridine (FUDR). L4 animals were incubated on NGM plates with OP50 and 50 μg/mL of FUDR supplementation at 20°C for 18–24 hr.S. aureus plates for 24 hr. 10 animals were harvested for each genotype. The animals were washed three times with ice cold M9 buffer with 0.1% trion X-100. After washing, the worms were resuspended in 300 μl M9 buffer with 0.1% trion X-100. An aliquot of the buffer was plated on NAL containing TSA plates to quantify external bacterial counts. Animals were then homogenized by a motor-driven pestle grinder. The homogenates were diluted and plated on NAL containing TSA plates. CFU from the pre-homogenized samples were subtracted from the CFU value after homogenization to calculate the CFU per animal values.Chemically sterilized young adults were infected with full lawn Worms were allowed to grow to young adult stage at 20°C on standard NGM plates with OP50. For each genotype, 120 young adults were then transferred to NGM plates with OP50 supplemented with 10 μM of FUDR. The cultures were continued at either 20°C or 25°C. Survival was monitored at least three times per week. Worms which did not respond to gentle touch by a worm pick were scored as dead and were removed from the plates. Animals that crawled off the plate or have ruptured vulva phenotypes were censored. All lifespan experiments were done three times independently unless otherwise stated. Genotypes were concealed during the lifespan experiments in order to eliminate any investigator-induced bias.C. elegans samples used for PCRs and RNA sequencing were prepared in the following way. Animals were allowed to grow at 20°C on NGM plates seeded with OP50 to age synchronized young adults. After washing with M9 buffer, they were transferred to 10 cm TSA plates supplemented with 10 μg/mL NAL carrying either S. aureus or heat-inactivated OP50 bacteria. TSA plates with heat-inactivated OP50 serve as the non-infection control here. The E. coli culture was concentrated 10 times before seeding on the plates. 500 μl of S. aureus or heat-inactivated E. coli OP50 were spread on the TSA plates, which were then incubated at 37°C for 6 hr. C. elegans worms were then transferred to the plates after the plates had equilibrated to room temperature. The animals were incubated on the plates at 25°C. After 4 hr of infection, the worms were washed with M9 buffer for two times before lysis with QIAzol Lysis Reagent (Qiagen).C. elegans and mammalian cells were lyzed with QIAzol Lysis Reagent. RNA was extracted using chloroform extraction method. The samples were then purified using RNeasy Mini Kit (Qiagen). Purity and concentration of the RNA samples were assessed using a NanoDrop 2000c (peqLab) equipment. cDNA synthesis was performed using iScript cDNA synthesis kit (Bio-Rad). Standard protocols provided by the manufacturers were followed for all mentioned commercial kits.prg-2, gyg-1 and tos-1 segments. The primer design for tos-1 is based on a recent publication (DreamTaq DNA Polymerases (ThermoFisher) was used to amplify the lication . PCR reasnb-1 was used for internal normalization for C. elegans targets. GAPDH or RPL13A were used for internal normalization for human targets. GAPDH was used for internal normalization of mouse targets. Relative expression levels were calculated using the comparative CT method. Special attention was paid to ensure that the primer sets detect all annotated isoforms. For primer sequences and binding locations, please refer to Power SYBR Green master mix (Applied Biosystems) was used for qPCR experiments. A JANUS automated workstation (PerkinElmer) was used for pipetting the reagents and cDNA samples into a 384 well plate. Thermal cycling was performed using a ViiA7 384 Real- Time PCR System machine (Applied Biosystems). rnp-6(dh1127), infected N2 and infected rnp-6(dh1127) in each biological replicate, three biological replicates in total) were pooled for quantification using the KAPA Library Quantification kit (Peqlab) and the 7900HT Sequence Detection System (Applied Biosystems). The libraries were then sequenced on one lane of an Illumina HiSeq4000 sequencing system using a paired end 2 × 75 nt sequencing protocol. For data analysis, Wormbase genome (WBcel235_89) was used for alignment of the reads. This was performed with the Hisat version 2.0.4 software. Differentially expressed genes (DEGs)  between different samples were identified using the stringtie version 1.3.0, followed by Cufflinks version 2.2. The DAVID  database was used for enrichment and Gene Ontology (GO) analysis. Cufflinks, KISSDE and SAJR were used for the splicing analysis. q value/p adjusted <0.05 is considered to be significant.1 μg of total RNA was used per sample for library preparation. We used the protocol of Illumina TruSeq stranded RiboZero. After purification and validation , total of 12 libraries  and PhosSTOP (Roche) and snap frozen in liquid nitrogen. The thawed samples were lyzed using Bioruptor Sonication System (Diagenode). For mammalian cell samples, the cells were first washed with PBS and then lyzed with RIPA buffer supplemented with cOmplete Protease Inhibitor (Roche) and PhosSTOP (Roche). Cell debris was removed by centrifugation. Protein concentration was estimated using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein samples were then heated to 95°C for 10 min in Laemmli buffer with 0.8% 2-mercaptoethanol in order to denature the proteins. The samples were sonicated in a sonicating water bath for 10 min if there is excessive DNA, which obscures the subsequent loading process. Samples were loaded on 4–15% MiniPROTEAN TGXTM Precast Protein Gels (Bio-Rad), and electrophoresis was performed at constant voltage of 200V for around 40 min. After separation, the proteins were transferred to PVDF membranes using Trans-Blot TurboTM Transfer System (BioRad). 5% Bovine serum albumin (BSA) or 5% milk in Tris-buffered Saline and Tween20 (TBST) were used for blocking of the membranes. After antibody incubations and washing with TBST buffer, imaging of the membranes was performed with ChemiDoc Imager (BioRad). Western Lightning Plus Enhanced Chemiluminescence Substrate (PerkinElmer) was used as the chemiluminescence reagent. A list of antibodies is provided in For Worms expressing HA::RNP-6, FLAG-SFA-1 or both were harvested, and proteins were extracted using the Bioruptor method. We used buffer containing 0.5% NP40, 150 mM NaCL and 50 mM Tris pH 7.4 supplemented with cOmplete Protease Inhibitor (Roche) and PhosSTOP (Roche) for immunoprecipitation. Flag immunoprecipitation was performed using Dynabeads Protein G (ThermoFisher Scientific) and FLAG M2 mouse monoclonal antibody (Sigma), following manufacturer’s protocols. Proteins were eluted from the beads by boiling with Laemmli buffer.E. coli HT115 and E. coli OP50 xu363. The HT115 bacteria were from the Vidal or Ahringer library. OP50 xu363 bacteria were transformed with dsRNA expression plasmids, which were extracted from the respective HT115 bacterial strains. The bacteria were grown in LB medium supplemented 100 μg/mL ampicillin at 37°C overnight with gentle shaking. The culture was spread on RNAi plates, which are NGM plates with addition of 100 μg/mL ampicillin and 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The RNAi bacteria were allowed to grow on the plates at room temperature for at least 12 hr. RNAi was initiated by letting the animals to feed on the desired RNAi bacteria. For infection survival experiments, rnp-6 RNAi was initiated around 30 hr before infection in order to prevent developmental abnormalities.Two different strains of RNAi competent bacterial strains were used in this study, namely siRNA against PUF60 and non-targeting siRNA were purchased from Dharmacon . Lipofectamine 3000 (ThermoFisher Scientific) was used as the transfection reagent for both siRNA and plasmids in HeLa cells. Lipofectamine RNAiMAX was used for siRNA transfections in RAW264.7 cells. For all transfection experiments, we followed the manufacturers’ protocols.S. aureus (MW2) was used for infection at multiplicity of infection (MOI) 100. Late logarithmic phase grown bacterial S. aureus cultures were diluted in DMEM with 10% FCS to the designated MOI. Cells were washed with PBS and then incubated with the bacterial suspension for 10 min at room temperature and another 30 min at 37°C with 5% CO2 in a humid atmosphere to allow internalization of the bacteria. After that, the infection medium was removed, and the residual extracellular bacteria were removed by washing with PBS and incubation with DMEM medium with 10% FCS and 50 μg/ml gentamicin for 1 hr. Lower concentration of gentamicin (10 μg/ml) was used for continued culture after the initial 1 hr incubation.To quantify the internalized bacteria, the cells were lyzed with PBS containing 0.3% of Triton X-100 at room temperature for 5 min to release intracellular bacteria. Amounts of bacteria were determined by serial dilution and plating on TSA plates.Cell viability was assayed by trypan blue exclusion method. At the indicated time points, infected HeLa cells were first washed with PBS to remove any dead cells in suspension. The cells were then dissociated by incubation with 0.5% trypsin. Cells were then mixed with trypan blue solution. Live cells which do not uptake trypan blue were counted using a hemocytometer.C. elegans strains, or any other resources should be directed to and will be fulfilled by the Lead Contact, Adam Antebi (Aantebi@age.mpg.de).Requests for reagents,  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Caenorhabditis elegans that might be conserved in other animals.The authors identified a splicing factor RNP-6/PUF60 whose activity suppresses immunity, but also promotes longevity, suggesting a trade-off between these two processes. The authors reveal the role of specific components of the splicing machinery in the modulation of immunity, which is important because the role of splicing in immunity is still poorly characterized. The merit of this article is that it illuminates a novel aspect of innate immune regulation in the nematode Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Orane Visvikis (Reviewer #1).Thank you for submitting your work entitled "Evolutionarily Conserved Regulation of Immunity by the Splicing Factor RNP-6/PUF60" for consideration by eLife. Nevertheless, they feel that there are too many requested work to allow a revision in two months. The consensus was therefore to reject the paper but to allow resubmission. If you decide to submit a revised version, it will be considered as a new submission but will likely be treated by the same reviewers. The decision 'Reject but allowing resubmission' gives you more time to revise the manuscript. We therefore encourage you to submit a new revised version of your manuscript that carefully addresses the point raised by the reviewers. If you feel that you cannot address the reviewer's comments, it is also possible to transfer the manuscript to another journal.Our decision has been reached after consultation between the reviewers. As you can see from the comments, the reviewers were rather positive about the manuscript and the possibility to publish it in Reviewer #1:C. elegans model and mammalian cell lines to elegantly unravel the evolutionary conserved immune function of the splicing factor RNP6/PUF60. By combining numerous and complementary approaches , the authors provide strong evidence of the repressive immune function of RNP-6/PUF60 in C. elegans, relying both on control of RNA splicing and gene expression, the latter lying upstream the pmk-1/p38 pathway. They also provide evidence that another splicing factor, namely SFA-1, work with RNP-6 to mediate host defense, underlying the importance of the RNA splicing function innate immunity. Finally, they show that PUF60 expression is repressed during infection of mammalian cell lines, which increases pro-inflammatory cytokines expression and promotes survival of infected cells. Overall, this well-conducted study reveals a novel function of PUF60 and contribute to enlarge the knowledge of a field in expansion. I would recommend this article for publication in eLife.In this article, Kew et al. have used rnp-6 RNAi was apparently performed using plates with partial lawn of S. aureus as the authors state to confirm this result with a survival assay using full lawn plates (subsection “The activity of RNP-6 impairs immunity”). Is the survival assay with partial lawn of S. aureus their standard assay ? This need to be clarified in the next and in the Materials and methods. Indeed, it is not clear how the experiments with rnp-6(dh1127) and rnp-6(dh1125) in Figure 1H  and Figure 1—figure supplement 1I, J and K, or with the overexpressing OE strains  were performed. Indeed, if based on the full lawn assay, the authors can conclude that the rnp-6 RNAi phenotype, which display extend survival compared to control, is not a difference in bacterial exposure, it is not clear whether the phenotypes of rpn-6 G281D mutants or of the OE strains, which display enhanced susceptibility to pathogens (Esp) compared to control, are due to reduced immune response or reduced aversive behavior  compared to N2. Full lawn experiment should be performed in order to conclude on the immune role of rnp-6. If partial lawn have been performed, the authors need to compare the bacterial lawn occupancy of RNP-6D281R mutants and the OE strains with N2. The author will be able to conclude on the immune regulatory function of RNP-6 only if RNP-6D281R mutant and N2 have the same avoidance behavior (and been exposed in a similar way to S. aureus).1) A major concern of this study relays on how survival assay were performed  which is not clear and may be of critical importance for the conclusion. Figure 4A: this survival assay testing 2) As a splicing factor, rpn-6 affects RNA splicing. I believe it would improve the manuscript to specify how primer pairs have been designed so that qPCR experiments reveal an actual change in gene expression and not a change in RNA splicing .pmk-1 and rnp-6 genetic experiment are made in animals infected by S. aureus, the authors analyze the effect RNP6 on PMK-1 phosphorylation either in resting condition (RNAi) or during infection by P. aeruginosa. It would greatly increase the quality of the conclusions to show 1) the level of P-PMK1 in N2i and rnp-6i animals infected by S. aureus; 2) the level of P-PMK1 in N2, rnp-6(dh1127) and RNP6 OE strain in the context of S. aureus infection. In addition, if anti-pmk1  can be obtained, it would be good to blot total pmk-1 to robustly show that variation of P-pmk-1 are really changes in signaling rather a change in total pmk-1 protein level.3) Figure 5GH and Figure 6H: condition of these experiments are rather surprising. While most of the 4) I believe the author should cite the work of the Sasakawa group  who had previously identified the Shigella NEL effector IpaH9.8 to bind RNA splicing factor U2AF35 to modulate host immune response. Although IpaH9.8 had later been shown to also target NEMO to control NF-κB and host immune response , it was shown that U2AF35 siRNA treatment reduces pro-inflammatory cytokines in Shigella-infected cells HeLa . Although this paper show opposite results in term of cytokine expression, it is a first evidence that modulation of a splicing factor, which is related to PUF60, during bacterial infection impacts immune responses, which reinforce the findings of Kew et al.Reviewer #2:C. elegans that include experiments showing similar interactions in human cells. The role of RNA splicing in stress responses of animal cells is poorly studied. This study identifies a conserved RNA binding protein that plays important roles in stress responses of C. elegans and human cells. Precise molecular mechanism have not been revealed, but the role of rnp-6 and splicing in immune response are convincingly demonstrated. Furthermore, the experiments are well conceived and reported. The genetic controls are particularly robust.This is a very solid investigation of how splicing factors influence stress and longevity signaling in 1) Stress survival and longevity data – These assays can be notoriously variable between trials. Therefore, reporting of data from at least three independent trials is customary for these types of studies. The comparisons should be analyzes as individual trials as well as combined. These data can be provided in supporting files.ret-1 reporter system. It appears that the GFP and RFP are largely expressed is different tissues  and that the changes in ratio are largely attributed to changes in only GFP versus a true shift from one to the other. The authors should include data for changes in GFP and RFP in addition to the rations. Furthermore, controls for total expression of the reporter would be needed to be sure that these effects are not a reflection of changes in total expression in certain tissues, versus a true shift in splicing within common tissues.2) I have concerns over the use of the 3) Figures 5G-H and 6H – quantification of the representative data for Figure 5H is missing. Also, it is customary to include blots for total PMK-1 to determine if the effects are at the total level or protein modification level.Reviewer #3:C. elegans and extend their study to a mammalian cell culture model. Because of my expertise, I restrict my comments on the experiments to those using C. elegans.Kew et al. investigate the link between splicing and the immunity and longevity in S. aureus infection in wildtype animals are associated with aldicarb resistance. As alternative splicing is tissue as well as stage specific (e.g. neuron-specific isoforms for many genes), this raises the possibility that the results are due to a differential effect of infection on transcription in a single tissue . The same applies for the effect seen in the rnp-6 mutant; changes in transcription within one tissue, or even subtle changes in developmental stage, would give the same result as alterations in splicing across tissues in an age-synchronized population.1) While the results are intriguing and would be of broad interest, I was concerned by Figure 3F, as in the infected sample, there are clearly larvae hatched inside that have a high GFP level and low mCherry level. This is a real problem. It indicates that (i) the experiments are not properly conducted (ii) the results involving differential splicing could be a result of differences that exist between different stages. Even if the worms shown were exceptional, it is notable that a significant number of the 83 genes alternatively spliced upon ret-1 was not identified in the sequencing-based analysis? It is unfortunate that the authors did not generated this type of reporter for at least one of the genes they identified as undergoing differential splicing.2) Given the apparent strong effect on the ret reporter, which presumably should reflect endogenous splicing patterns, how is it that S. aureus infection or the rnp-6 G281D mutation", what is the criterion for "significant", especially if one looks at the last example, cebp-1, for which there is a stark difference regarding retention of the last intron between wild-type and rnp-6 samples. [typo: S. aurues in figure]. This is particularly relevant given the known links between cebp-1, its 3' UTR (pubmed/28673818), pmk-1 (pubmed/27927209) and innate immunity.3) Figure 5—figure supplement 1 shows sequencing reads coverage for several genes. If the figure is only matching pairs for their paired end 2x75nt sequencing, which it should be for this type of analysis, the level of intronic signal for several genes is unexpected. So, when they write, "The splicing patterns of these genes are not significantly affected by 4) To understand the basis of altered resistance, it is standard in the field to perform simple tests to assay the extent of bacterial colonization.nlp-30, there is the same fold-change after infection in mutant and wild-type, so the induction is not rnp-6 dependent. One therefore wonders whether their strategy for gene selection  is entirely appropriate.5) Interpretation of Figure 2E is complicated by the fact that the constitutive expression level of at least one of the genes under study differs between wild-type and mutant worms. Thus, for eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Tadatsugu Taniguchi as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Orane Visvikis (Reviewer #1).Thank you for submitting your article "Evolutionarily conserved regulation of immunity by the splicing factor RNP-6/PUF60" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:Caenorhabditis elegans that might be conserved in other animals.The authors identified a splicing factor RNP-6/PUF60 whose activity suppresses immunity, but promotes longevity, suggesting a trade-off between these two processes. The authors reveal the role of specific components of the splicing machinery in the modulation of immunity, which is important because the role of splicing in immunity is still poorly characterized. The merit of this article is that it illuminates a novel aspect of innate immune regulation in the nematode Essential revisions:S. aureus and P. aeruginosa infection. "RNP-6 exerts its inhibitory effects on immunity by suppressing the activity of PMK-1" (L 305-306) is over-stated as the authors do not have proof that rnp-6 impact the activation (phosphorylation) of PMK-1 during infection. Indeed:1) Despite the additional data provided, it is still not clear how RNP-6 impacts the PMK-1 pathway during rnp-6 OE strain or rnp-6 G281D mutant on the phospho-level of PMK-1 during S. aureus infection .– The authors find no effect of rnp-6 RNAi reduces P-PMK-1 during S. aureus infection, but this result is rather questionable considering the western-blot shown . There are huge differences in total protein levels as revealed by the loading control (actin), but these differences are not as strong in total PMK-1. This either suggest that 1) that the level of total PMK-1 varies, or 2) the experiment is not well conducted.– The authors state that – Figure 5—figure supplement 1C: control of total PMK-1 has not been provided. In addition, the quantification of this blot clearly indicates that there is a huge variation between experiments, and that P-PMK1 variation between conditions is not statistically significant.I don't think this affect the quality of the manuscript, but the author should be accurate in their interpretation, and the their conclusion modified accordingly . Similarly, the text should be changed in the Discussion "How altering splicing can lead to activation of PMK-1.…".rnp-6 G281D mutant compared to WT, which still leaves a doubt on a potential lack of avoidance behavior of the rnp-6 mutant . Fortunately, the author also provide full lawn survival experiments as requested and found the rnp-6 G281D and rnp-6 OE strains to also display Esp compared to WT animals, indicating that these strains indeed have an immune defect. The authors keep describing this immune mechanism as "resistance" which I believe is not an appropriate term, especially in this new version of the manuscript where the authors, to answer reviewer 3, now assess the bacterial burden. Resistance, as defined by the capacity to limit bacterial burden, is opposed to tolerance, defined as ability to limit health impact of a given pathogen burden . In this type of survival assays, where animals are constantly exposed and fed with pathogenic bacteria, it is difficult to assess the resistance capacity. Assessing resistance capacity is easier to detect when animals are transfer onto non-pathogenic food . Accordingly, the colonization assays, performed on animals constantly fed for 24h on S. aureus, show similar bacterial burden between control and rnp-6G281D mutant, rnp-6 OE strains or rnp-6 RNAi treated animals . Rather, these full lawn survival assays, controlled by similar colonization assays, allow to assess the tolerance capacity of the strains. Together, these results indicate that the immune function of rnp-6 relies on a tolerance mechanism. This should be indicated in the text which, I believe, would improve the quality of the article. However, the term resistance, which has been repeatedly found though-out the manuscript, is miss-used and should be replaced by i.e.: "decreased sensitivity" or "increased survival" and only when appropriate, by tolerance. Whether rnp-6 has also resistance immune branch should not be excluded, as it was difficult to assess in this experimental setting. Please also correct : "rnp-6 RNAi did not affect S. aureus bacterial burden, suggesting the resistance is caused by independent mechanisms".2) As requested, the authors have performed bacterial lawn occupancy experiments and found it is increased in ret-1 reporter. As mCherry and GFP have different half-lives, changes in fluorescence intensity under a pathological situation need not reflect a change in splicing. The magnitude of the change of GFP expression is considerably higher than that of mCherry; it seems that a change in splicing is not the most important effect here. Further, according to the dot plot, the GFP level for the vast majority of "Infected rnp-6" worms is below that for the majority of "rnp-6(dh1127)" worms, so the photomicrograph is totally unrepresentative and very misleading.3) There are still problems with the data presented in Figure 3 and in Figure 3—figure supplement 1. Indeed, there is no demonstration of a correlation between splicing as measured by PCR and using reporter genes. Thus, they neither "generate this type of reporter for at least one of the genes they identified as undergoing differential splicing", nor do they provide PCR evidence to support their observations with the P. aeruginosa infection condition in multiple biological replicates, although this did not reach statistical significance due variability of phospho-PMK-1 levels between replicates" are not appropriate. The results are simply not statistically significant and no inferences can be made.4) Statements like, "By contrast, we observed that the infection sensitive G281D substitution mutant of RNP-6 had a consistently suppressed phosphorylation levels of PMK-1 under  [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Reviewer #1:S. aureus as the authors state to confirm this result with a survival assay using full lawn plates (subsection “The activity of RNP-6 impairs immunity”). Is the survival assay with partial lawn of S. aureus their standard assay ? This need to be clarified in the next and in the Materials and methods. Indeed, it is not clear how the experiments with rnp-6 (dh1127) and rnp-6 (dh1125) in Figure 1H  and Figure 1—figure supplement 1I, J and K, or with the overexpressing OE strains  were performed. Indeed, if based on the full lawn assay, the authors can conclude that the rnp-6 RNAi phenotype, which display extend survival compared to control, is not a difference in bacterial exposure, it is not clear whether the phenotypes of rpn-6 G281D mutants or of the OE strains, which display enhanced susceptibility to pathogens (Esp) compared to control, are due to reduced immune response or reduced aversive behavior  compared to N2. Full lawn experiment should be performed in order to conclude on the immune role of rnp-6. If partial lawn have been performed, the authors need to compare the bacterial lawn occupancy of RNP-6D281R mutants and the OE strains with N2. The author will be able to conclude on the immune regulatory function of RNP-6 only if RNP-6D281R mutant and N2 have the same avoidance behavior (and been exposed in a similar way to S. aureus).[…] 1) A major concern of this study relays on how survival assay were performed  which is not clear and may be of critical importance for the conclusion. Figure 4A: this survival assay testing rnp-6 RNAi was apparently performed using plates with partial lawn of S. aureus plates.Although we used partial lawn for our standard assays, we also performed the infection survival experiments using full lawn rnp-6(dh1127) mutants and RNP-6 overexpressing animals exhibit Esp on full lawn S. aureus infection plates, indicating that the Esp phenotype cannot be explained by differential exposure to bacteria.We also compared the lawn occupancy of rnp-6(dh1127) mutants to N2. We observed a general increase of lawn occupancy in all strains during the course of S. aureus infection, which is probably due to physical inactivity caused by advanced infection. Interestingly, rnp6(1127) mutants had a higher lawn occupancy compared to N2. This could be explained by the more rapid progression of infection in this strain. Another infection sensitive mutant strain, pmk-1(km25), displayed a similar phenotype, suggesting higher lawn occupancy may be a common phenotype of infection sensitive strains as their physical activity declines faster due to quicker progression of the infection process.Clearly, both rnp-6(dh1127) and RNP-6 overexpressing animals.All in all, we conclude that differential exposure to bacteria is not the cause of the Esp phenotype of 2) As a splicing factor, rpn-6 affects RNA splicing. I believe it would improve the manuscript to specify how primer pairs have been designed so that qPCR experiments reveal an actual change in gene expression and not a change in RNA splicing .Special attention was paid to ensure that the qPCR primer binding sites are located in regions which are included in all annotated isoforms. Therefore, the qPCR primers should detect all annotated isoforms and reflect an actual change in gene expression. We provide visualization of all qPCR primer binding sites in the Supplementary file 1. Notes have also been added to the Materials and methods section to clarify this issue.Y43F8B.9 was mislabeled as frm-7. We corrected the corresponding figure in Figure 2—figure supplement 1. This does not affect the conclusion of the study.While we were preparing the lists and the schematics of the primers, we found that the primers for S. aureus, the authors analyze the effect RNP6 on PMK-1 phosphorylation either in resting condition (RNAi) or during infection by P. aeruginosa. It would greatly increase the quality of the conclusions to show 1) the level of P-PMK1 in N2i and rnp-6i animals infected by S. aureus; 2) the level of P-PMK1 in N2, rnp-6(dh1127) and RNP6 OE strain in the context of S. aureus infection. In addition, if anti-pmk1  can be obtained, it would be good to blot total pmk-1 to robustly show that variation of P-pmk-1 are really changes in signaling rather a change in total pmk-1 protein level.3) Figures 5G-H and 6H: condition of these experiments are rather surprising. While most of the pmk-1 and rnp-6 genetic experiment are made in animals infected by rnp-6 and sfa-1 RNAi Western blots .We added the total PMK-1 blots for the S. aureus infected animals.We also measured the relative levels of phospho-PMK-1 in S. aureus on the levels of phosphoPMK-1. However, we observed a reduction of phospho-PMK-1 upon S. aureus infection in rnp-6 RNAi treated animals. It seems that the PMK-1 activity is affected by S. aureus in an RNP-6 dependent manner.Generally, we did not observe any significant effects of 4) I believe the author should cite the work of the Sasakawa group  who had previously identified the Shigella NEL effector IpaH9.8 to bind RNA splicing factor U2AF35 to modulate host immune response. Although IpaH9.8 had later been shown to also target NEMO to control NF-κB and host immune response , it was shown that U2AF35 siRNA treatment reduces pro-inflammatory cytokines in Shigella-infected cells HeLa . Although this paper show opposite results in term of cytokine expression, it is a first evidence that modulation of a splicing factor, which is related to PUF60, during bacterial infection impacts immune responses, which reinforce the findings of Kew et al.The reference has been added.Reviewer #2:[…] 1) Stress survival and longevity data – These assays can be notoriously variable between trials. Therefore, reporting of data from at least three independent trials is customary for these types of studies. The comparisons should be analyzes as individual trials as well as combined. These data can be provided in supporting files.We provide the p values, number of animals analysed and median survival of all replicates of all infection survival and lifespan experiments in Supplementary file 3.2) I have concerns over the use of the ret-1 reporter system. It appears that the GFP and RFP are largely expressed is different tissues  and that the changes in ratio are largely attributed to changes in only GFP versus a true shift from one to the other. The authors should include data for changes in GFP and RFP in addition to the rations. Furthermore, controls for total expression of the reporter would be needed to be sure that these effects are not a reflection of changes in total expression in certain tissues, versus a true shift in splicing within common tissues.C. elegans. Therefore, the quantification reflects a shift of abundance of GFP and mCherry in the same tissue, not a comparison overall.First, we quantified the GFP and mCherry signals from the anterior gut as this is the relevant tissue for bacterial infection in ret-1 reporter experiment using a new protocol as suggested by reviewer 3 (with addition of FUDR treatment). The data shown below is the new data.As the reviewer requested, we provide the quantification of GFP and mCherry separately. Please note that we repeated the infection S. aureus infection clearly reduced the GFP to mCherry ratio, and this effect was partially suppressed by the rnp-6(dh1127) mutation. GFP intensity decreased drastically in infected N2 but only moderately in infected rnp-6(dh1127) animals. mCherry expression was induced by S. aureus infection in both WT and rnp-6(dh1127) animals. Although we observed a trend of lower mCherry signal in infected rnp-6(dh1127) mutants (mean=11.944) compared to infected N2 (mean=12.499), the effect does not reach statistical significance.Similar results can also be seen in rnp-6 RNAi treated animals. knocking-down rnp-6 reduced GFP to mCherry ratio, which mimics the situation of infection. rnp-6 RNAi not only reduced the GFP intensity, but also induced the mCherry signal.rnp-6 reduction result in a similar shift of the ret-1 splicing reporter, which is caused by a decrease of GFP expression and a concomitant increase of mCherry.Based on these data, we conclude that both bacterial infection and 3) Figures 5G-H and 6H – quantification of the representative data for 5H is missing. Also, it is customary to include blots for total PMK-1 to determine if the effects are at the total level or protein modification level.P. aeruginosa infected animals. Although we observed a consistent trend of PMK-1 activation upon P. aeruginosa infection in three independent replicates, the relative levels of activation were highly variable and did not reach statistical significance. We moved this data to the figure supplement.We did the quantification of phospho-PMK-1 for rnp-6 and sfa-1 RNAi Western blots .Further, we added the total PMK-1 blots for the Reviewer #3:C. elegans and extend their study to a mammalian cell culture model. Because of my expertise, I restrict my comments on the experiments to those using C. elegans.Kew et al. investigate the link between splicing and the immunity and longevity in S. aureus infection in wildtype animals are associated with aldicarb resistance. As alternative splicing is tissue as well as stage specific (e.g. neuron-specific isoforms for many genes), this raises the possibility that the results are due to a differential effect of infection on transcription in a single tissue . The same applies for the effect seen in the rnp-6 mutant; changes in transcription within one tissue, or even subtle changes in developmental stage, would give the same result as alterations in splicing across tissues in an age-synchronized population.1) While the results are intriguing and would be of broad interest, I was concerned by Figure 3F, as in the infected sample, there are clearly larvae hatched inside that have a high GFP level and low mCherry level. This is a real problem. It indicates that (i) the experiments are not properly conducted (ii) the results involving differential splicing could be a result of differences that exist between different stages. Even if the worms shown were exceptional, it is notable that a significant number of the 83 genes alternatively spliced upon rnp-6(dh1127) has a slight developmental delay. We did the egg lay sequentially to ensure the synchronization of different strains. Animals were also examined visibly before experiments to make sure that they are well-synchronized.The worms were synchronized with special care, as prg-2, gyg-1 and tos-1 on day2 adult animals.To further exclude the possibility of the effects of staging, we repeated the splicing PCR of gyg-1, for which day 2 rnp-6(dh1127) animals responded to infection comparably to N2 animals. In day 1 young adults, the gyg-1 splicing change induced by S. aureus infection is partially suppressed in rnp-6(dh1127) animals. Because of the special attention given for the synchronization of the animals and the reproducibility of the results in another life stage (Day 2 adulthood), the splicing changes we observed are unlikely to be caused by lack of synchronization or subtle changes in developmental stage, but rather to be the results of infection and/or the rnp-6 mutation.We also repeated the ret-1 reporter experiment with FUDR sterilized animals, which eliminates hatching of larvae, and we obtained similar results to the experiments performed without FUDR. We quantified the signals from anterior gut as this is the relevant tissue for bacterial infection in C. elegans. See The splicing patterns of day2 adults are generally consistent with those of day 1 young adults, except for Given the tight staging, it seems less likely that tissue composition is different between genotypes. If there are tissue specific effects  it still would be due to splicing, and this is inherently interesting, but lies beyond the scope of the study.2) Given the apparent strong effect on the ret reporter, which presumably should reflect endogenous splicing patterns, how is it that ret-1 was not identified in the sequencing-based analysis? It is unfortunate that the authors did not generated this type of reporter for at least one of the genes they identified as undergoing differential splicing.ret-1 reporter imaging were done at different timepoints. We collected the RNA at post-infection 4 hours whereas the imaging of ret-1 reporter was done at post-infection 9 hours. The effects on ret-1 maybe a delayed response. To test this hypothesis, we repeated the ret-1 reporter imaging experiment at post-infection 4 hours.First, the RNA sequencing and the ret=1 reporter were very small compared to that of the later timepoint, which may be one of the reasons why ret-1 was not identified in the sequencing-based analysis. Also, the sequencing was done on RNA extracted from the whole animals which may complicate the analysis and average out any tissue specific effects of ret-1 splicing.At this earlier timepoint, the effects on the S. aureus infection or the rnp-6 G281D mutation", what is the criterion for "significant", especially if one looks at the last example, cebp-1, for which there is a stark difference regarding retention of the last intron between wild-type and rnp-6 samples. [typo: S. aurues in figure]. This is particularly relevant given the known links between cebp-1, its 3' UTR (pubmed/28673818), pmk-1 (pubmed/27927209) and innate immunity.3) Figure 5—figure supplement 1 shows sequencing reads coverage for several genes. If the figure is only matching pairs for their paired end 2x75nt sequencing, which it should be for this type of analysis, the level of intronic signal for several genes is unexpected. So, when they write, "The splicing patterns of these genes are not significantly affected by cebp-1. The retention of the last intron is variable between biological replicates. Thus, this is likely to be a noise instead of a true biological phenotype.q value/p adjusted < 0.05 is considered to be significant. The following are the additional replicates of the sequencing data of 4) To understand the basis of altered resistance, it is standard in the field to perform simple tests to assay the extent of bacterial colonization.rnp-6(dh1127), rnp-6 RNAi or overexpression on S. aureus colonization, suggesting that rnp-6 affects infection resistance through other mechanisms .We did not observe any observable effects of 5) Interpretation of Figure 2E is complicated by the fact that the constitutive expression level of at least one of the genes under study differs between wild-type and mutant worms. Thus, for nlp-30, there is the same fold-change after infection in mutant and wild-type, so the induction is not rnp-6 dependent. One therefore wonders whether their strategy for gene selection  is entirely appropriate.nlp-30 are lower in rnp-6(dh1127) mutants regardless of infection, suggesting that the expression of nlp-30 is dependent on rnp-6 under both basal and infection conditions. nlp-30 is still induced in infected rnp-6(dh1127) animals, suggesting that the mutation does not completely inhibit the induction of nlp-30 by infection. The mutated RNP-6 may still mediate the induction of nlp-30. Alternatively, there may be alternative and/or compensatory mechanisms to regulate the expression of nlp-30 in rnp-6(dh1127) animals. Nevertheless, the data supports that rnp-6 affects the expression of nlp-30, and also additional regulation exists.The relative levels of [Editors’ note: what follows is the authors’ response to the second round of review.]Essential revisions:S. aureus and P. aeruginosa infection. "RNP-6 exerts its inhibitory effects on immunity by suppressing the activity of PMK-1" (L 305-306) is over-stated as the authors do not have proof that rnp-6 impact the activation (phosphorylation) of PMK-1 during infection. Indeed:1) Despite the additional data provided, it is still not clear how RNP-6 impacts the PMK-1 pathway during S. aureus infection .– The authors find no effect of rnp-6 OE strain or rnp-6 G281D mutant on the phospho-level of PMK-1 during S. aureus infection, but this result is rather questionable considering the western-blot shown . There are huge differences in total protein levels as revealed by the loading control (actin), but these differences are not as strong in total PMK-1. This either suggest that 1) that the level of total PMK-1 varies, or 2) the experiment is not well conducted.– The authors state that rnp-6 RNAi reduces P-PMK-1 during – Figure 5—figure supplement 1C: control of total PMK-1 has not been provided. In addition, the quantification of this blot clearly indicates that there is a huge variation between experiments, and that P-PMK1 variation between conditions is not statistically significant.I don't think this affect the quality of the manuscript, but the author should be accurate in their interpretation, and the their conclusion modified accordingly . Similarly, the text should be changed in the Discussion "How altering splicing can lead to activation of PMK-1.…".pmk-1 is required for resistance to bacterial pathogens in C. elegans, and mutations or treatments that induce phosphorylation of PMK-1 lead to enhanced survival , whether bacterial pathogens actually induce phosphorylation of PMK-1 is debated.We consulted other experts from the field concerning pathogen dependent PMK-1 phosphorylation. Although it is well known that S. aureus does not activate PMK-1 phosphorylation , which we also confirmed. On the other hand, PA14 infection leads to high variability of phospho-PMK-1 levels , which is also consistent with our data. Conceivably these differences might reflect differences in timing, or culture conditions.rnp-6i treated animals, which indicate pmk-1 acts downstream of rnp-6.Therefore, the absence of PMK-1 phosphorylation upon infection is not inconsistent with previous findings, and does not compromise the results from survival epistasis and phosphoPMK-1 blots of We have modified the Discussion section according to the reviewers’ suggestions.S. aureus, show similar bacterial burden between control and rnp-6G281D mutant, rnp-6 OE strains or rnp-6 RNAi treated animals . Rather, these full lawn survival assays, controlled by similar colonization assays, allow to assess the tolerance capacity of the strains. Together, these results indicate that the immune function of rnp-6 relies on a tolerance mechanism. This should be indicated in the text which, I believe, would improve the quality of the article. However, the term resistance, which has been repeatedly found though-out the manuscript, is miss-used and should be replaced by i.e.: "decreased sensitivity" or "increased survival" and only when appropriate, by tolerance. Whether rnp-6 has also resistance immune branch should not be excluded, as it was difficult to assess in this experimental setting. Please also correct : "rnp-6 RNAi did not affect S. aureus bacterial burden, suggesting the resistance is caused by independent mechanisms".2) As requested, the authors have performed bacterial lawn occupancy experiments and found it is increased in rnp-6 G281D mutant compared to WT, which still leaves a doubt on a potential lack of avoidance behavior of the rnp-6 mutant . Fortunately, the author also provide full lawn survival experiments as requested and found the rnp-6 G281D and rnp-6 OE strains to also display Esp compared to WT animals, indicating that these strains indeed have an immune defect. The authors keep describing this immune mechanism as "resistance" which I believe is not an appropriate term, especially in this new version of the manuscript where the authors, to answer reviewer 3, now assess the bacterial burden. Resistance, as defined by the capacity to limit bacterial burden, is opposed to tolerance, defined as ability to limit health impact of a given pathogen burden . In this type of survival assays, where animals are constantly exposed and fed with pathogenic bacteria, it is difficult to assess the resistance capacity. Assessing resistance capacity is easier to detect when animals are transfer onto non-pathogenic food . Accordingly, the colonization assays, performed on animals constantly fed for 24h on We thank the reviewers for pointing out the difference between resistance and tolerance/survival. We have modified the manuscript by omitting the word pathogen resistance, as suggested.3) There are still problems with the data presented in Figure 3 and in Figure 3—figure supplement 1. Indeed, there is no demonstration of a correlation between splicing as measured by PCR and using reporter genes. Thus, they neither "generate this type of reporter for at least one of the genes they identified as undergoing differential splicing", nor do they provide PCR evidence to support their observations with the ret-1 reporter. As mCherry and GFP have different half-lives, changes in fluorescence intensity under a pathological situation need not reflect a change in splicing. The magnitude of the change of GFP expression is considerably higher than that of mCherry; it seems that a change in splicing is not the most important effect here. Further, according to the dot plot, the GFP level for the vast majority of "Infected rnp-6" worms is below that for the majority of "rnp-6(dh1127)" worms, so the photomicrograph is totally unrepresentative and very misleading.Because this reporter had been previously published as reflecting splicing , we took it at face value, but are grateful that the reviewers pointed out possible different interpretations of this reporter.ret-1. .To address the reviewers’ concerns, we performed RT-PCR to assess the relative abundance of the two isoforms of rnp-6 RNAi and S. aureus infection induced ret-1 exon 5First, we confirmed that both ret-1 transcript.skipping, although the effects seem to be less dramatic as indicated by the reporter system, suggesting that the splicing reporter might not truly reflect the splicing of the endogenous rnp-6(dh1127) mutation did not inhibit the infection induced splicing of ret-1. Rather, the basal level of exon 5 skipping is slightly elevated in the rnp-6 mutants. These observations were not reflected by the splicing reporter.Furthermore, we observed that the ret-1 transcript. And finally, the signals from the reporter might reflect other processes, including mRNA and/or protein stability, translation and protein degradation.Multiple explanations could potentially account for the discrepancy of the two methods. First, we focused on a specific tissue (anterior gut) for the splicing reporter experiments whereas RT-PCR reflects the splicing events averaged over the whole animals. Second, some elements, such as splicing enhancers, may not be included into the reporter, which can lead to different splicing patterns compared to endogenous ret-1 splicing reporter does not accurately reflect the splicing of ret-1 endogenous transcript. Therefore, we decided to remove the relevant data from the manuscript .We conclude that the rnp-6 RNAi affects the splicing (as measured by RTPCR) of prg-2, gyg-1 and tos-1, which we identified to show rnp-6 G281D dependence upon infection  .To replace Figure 4C, we checked how rnp-6 RNAi affected prg-2 and tos-1 splicing, but had no observable effect on gyg-1. rnp-6 RNAi promoted the splicing of prg-2 intron 1, similar to the effect of S. aureus infection , but opposite to rnp-6 G281D mutant. On the other hand, rnp-6 RNAi inhibited the splicing of tos-1 intron 1, and this splicing pattern was similar to what we observed for the rnp-6 G281D mutant .We observed that rnp-6 G281D mutation inhibits infection induced prg-2 intron 1 splicing, whereas rnp-6 RNAi promotes the splicing event. In this regard, the rnp-6 G281D mutation resembles a gain-of function. However, both rnp-6 G281D and rnp-6 RNAi reduce the splicing of tos-1 intron 1. Therefore, the G281D mutation and rnp-6 RNAi result in partially opposite and also overlap splicing, similar to the results of transcript abundance of immune-genes . Nevertheless, these results confirm that rnp-6 RNAi affects splicing of the genes whose splicing is moderated by infection, strengthening the role of RNP-6 in infection induced splicing remodeling.In conclusion, the P. aeruginosa infection condition in multiple biological replicates, although this did not reach statistical significance due variability of phospho-PMK-1 levels between replicates" are not appropriate. The results are simply not statistically significant and no inferences can be made.4) Statements like, "By contrast, we observed that the infection sensitive G281D substitution mutant of RNP-6 had a consistently suppressed phosphorylation levels of PMK-1 under As discussed, phospho-PMK-1 levels upon infection can be very variable. We removed this data from the manuscript.References:Cao, X., and Aballay, A. 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